The ever-increasing interest in microanalysis, especially in the fields of cell biology, electronics, and materials science, have demanded specialized techniques which must provide both high spatial resolution and high sensitivity. The currently available techniques fall into two broad classes: the first involves the use of shorter wavelength radiation such as in electron or x-ray microscopy [D. F. Parsons, editor, Short Wavelength Microscopy, New York Academy of Sciences, New York 1978; D. Sayer, editor, X-ray Microscopy II Proceedings of the Second International Symposium, Springer-Verlag, New York, 1988]. The second class includes the various forms of scanning probe microscopy which include scanning tunneling microscopy (STM) as the best-known example [H. K. Wickramasinghe, editor, Scanned Probe Microscopy, American Institute of Physics, New York, 1992; Binning et al., Phys. Rev. Lett. 49: 57 (1982)]. Unfortunately, however, most of these techniques either involve radiation that is destructive to the sample itself or requires test circumstances that are not environmental conditions or are not ambient conditions in which the sample is found.
In contrast and contradistinction, microanalytical techniques, such as optical microscopy, provide the highly desirable and attractive features of low cost, high speed, reliability, versatility, accessibility, ease of use, and information contrast. Equally important, optical microscopy in the visible region of the spectrum is generally regarded as the most benign kind of electromagnetic radiation. In addition, optical microscopy does not require the sample to be subjected to vacuum; does not utilize sample-destructive electron beams as is the case with scanning electron microscopy; and is not limited to the use of conductive samples as is the case with scanning tunneling microscopy. Moreover, optical techniques can be interfaced with a wide variety of selective absorption and fluorescence probes such that many molecules and cellular structures can be observed. Unfortunately, the usual problem with visible light and optical microscopy is the limitation imposed by focusing with a lens; and resolution is thus the primary limitation of lens-based optical microscopy.
The resolution of all lens-based instruments is defined by the finite dimensions of a lens. The resolving power, or the minimum separation between two points which can be resolved (d.sub.min), can be approximated by the following equation: d.sub.min .about..lambda./2 where .lambda. is the wavelength of light. This limitation is the result of diffraction that takes place in the microscope because of the wave nature of light. Moreover, it should be noted that the resolution limit described above arises from the assumption that the image of an object is being detected in the "Far-Field", that is, a distance which is much greater than the wavelength of light employed (e.g., the wavelength of green light is ca. 500 nm). For these reasons, where the optical microscopy is so limited in resolution to no better than one-half of the wavelength of light being used (.lambda./2), such microscopy is termed "far-field" imaging or viewing.
An increasingly important and rapidly developing alternative to conventional lens-based optical microscopy is the "near-field" or lensless technique which provides superresolution imaging and spectroscopy. The term "superresolution" defines any means for optical imaging or spectroscopy that permits spatial resolution which exceeds the diffraction limitation caused by the wave nature of light; and provides a resolution which is greater than one-half the wavelength of the light actually being used. All superresolution near-field imaging and near-field scanning optical microscopy ("NSOM") is based on the fact that although light cannot be focused to a spot less than one-half the wavelength of light (.lambda./2), it can be directed through a device or article which reduces the size of the light energy to dimensions smaller than .lambda./2. A variety of size reducing probe articles have been proposed and apply to near-field imaging and to near-field scanning optical microscopy. These have come in three essential categories: devices that utilize apertures [Betzig, E. and J. K. Trautman, Science. 257: 189-195 (1992); Betzig et. al., Science 251: 1468 (1991); Lewis et al, Ultramicroscopy 13: 227 (1984); Pohl, et al., ,App. Phys. Lett. 44: 651 (1984); Betzig, et al., Biophys. J. 49: 269 (1986)]; those devices involving near-field scattering mechanisms [Pohl, et al., Proc. Soc. Photo-opt. Instru. Eng. 897: 94 (1988); Fischer, et al., Phys. Rev. Lett. 62: 458 (1989); Reddick, et al., Phys. Rev. B. 39: 767 (1989)]; and those methods dependent upon luminescence effects [Lieberman et al., Science 247: 59 (1990); Lewis, A and K. Lieherman, Nature 354: 214 (1991); U.S. Pat. No. 5,105,605].
The basic principle of near-field viewing and imaging is best illustrated by the aperture technique as is illustrated by prior art FIG. A. When light is directed through a sub-.lambda.-sized hole, the portion of energy that passes through the hole will at first be confined to the dimensions of the aperture. The exiting light being of subwavelength dimensions will then rapidly diffract in all directions; however, there will be a distinct region in the vicinity of the aperture called the "near-field" where the existing light beam retains the approximate dimensions of the hole. If this subwavelength light beam within the near-field region is used to raster scan the surface of an object, a two-dimensional image can be created in a serial fashion (one point at a time). Resolution far less than the conventional lens-based limit of about 200 nanometers (nm) is easily achieved; and frequently resolution on the order of 15-50 nanometers can be achieved using the NSOM technique.
It will be recognized and appreciated that the essential concept of superresolution near-field imaging was presented more than sixty years ago by E. H. Synge [Phil. Mag. 6: 356 (1928)]; regenerated about 40 years ago by J. A. O'Keefe [J. Opt. Soc. Am. 15: 359 (1956)]; and only validated in 1972 by an experiment which obtained .lambda./60 resolution by passing microwaves through a small aperture and scanning it over a surface [Ash, E. A. and G. Nicholls, Nature 237: 510 (1972)]. The near-field superresolution technique remained only of passing interest for some years thereafter [Lewis et al., Biophys. J. 11: 405a (1983); Lewis et al., Ultramicroscopy 13: 227 (1984); Pohl et al., Appl. Phys, Lett. 11: 651 (1984)]. It is only been in the last few years that near-field imaging and NSOM has become of primary interest as an alternative technique in optical microscopy [Lieberman et al., Science 247: 59 (1990); Kopelman et al., Microbeam Analysis, D. G. Howitt, editor, San Francisco Press Inc., 1991; Lewis, A. and K. Lieberman, Anal. Chem. 63: 625A (1991); Betzig, E. and J. K. Trautman, Science 257: 189 (1992); Harris et al., Applied Spectroscopy 48: 14A (1994); and the references cited within each of these individual publications].
The most favored approach to date for near-field imaging and NSOM has been the use of apertures and the development of aperture-containing probes which will overcome the technological difficulties imposed by the NSOM approach. It is recognized that the chief difficulty in near-field imaging and the NSOM technique lies primarily in the fabrication of a suitable aperture; and in the ability to position the aperture accurately near the surface of the object to be imaged and yet be close enough to the surface of the object that the subwavelength light beam remains collimated. The entire near-field region typically extends no further than the dimensions of the subwavelength aperture itself. Accordingly, to maintain consistency of light beam size and intensity of light energy, the actual distance between the surface of the object and the subwavelength-aperture must be held constant to within a few percent of the entire near-field region itself.
Several different kinds of subwavelength aperture-containing probes or articles have been developed and reported in the scientific literature. A first instance has taken the form of metal-coated glass micropipettes [Harootunian et al, Appl. Phys. Lett. 19: 674 (1986)]. These micropipettes were produced by heating and pulling apart small glass capillaries to yield an inner aperture diameter in the range of between 500-1000 Angstroms (A). The heat-pulled glass capillaries are then evaporation coated along the outside of the glass micropipette with a thin film of metal such as aluminum or chrome--thereby making the exterior surface completely opaque. Typically thereafter, a small optical fiber strand is inserted into the interior lumen of the coated glass pipette up to the very tip near the aperture; and a laser beam of light is fed into the pipette interior via the optical fiber. As a consequence, a tiny beam of light energy emerges from the uncoated hole at the tip of the micropipette. In this manner, a controlled subwavelength beam of light energy emanating from the tapered micropipette can be employed for near-field scanning optical microscopy [Betzig, E. and J. K. Trautman, Science 257: 189 (1992) and the references cited therein].
Another instance of the aperture technique is the use of a clear optical fiber tapered adiabatically to a tiny tip and subsequently coated with aluminum [Betzig et al., Science 251: 1468 (1991)]. Using this subwavelength apertured probe in combination with a light beam from an argon ion laser through an 80 nanometer aperture, near-field images with .lambda./43 resolution were obtained.
A third instance of aperture probes utilizes crystals of anthracene, dichloromethane, and tetracene to transform the aperture point from a passive source of subwavelength light into an active emitter of light. These articles employ the submicron tip of a metal-coated glass micropipette whose interior is filled with the molecular crystal. Incoming light photons propagate through the submicrometer portion of the pipette and become absorbed by the crystal. The crystal then actively emits the light beam through the aperture for near-field imaging [Kopelman et al., Microbeam Analysis (D. G. Howitt, editor), San Francisco Press, Inc., 1991].
A fourth instance utilizes a flat aperture consisting of a glass slide covered with a thick aluminum film containing small subwavelength dimension holes formed by metal-shadowing small latex spheres. [Fischer, U. Ch., J. Vac. Sci. Technol. B3: 3861 (1985)]. The problem of positioning the flat aperture probe in relation to the surface of a sample was resolved by placing a flat sample on the top of a spherical glass surface.
Despite these many recent developments and innovations reported in the scientific literature, near-field imaging and near-field scanning optical microscopy remains a difficult and burdensome technique in practice. Each of the presently known aperture devices for producing subwavelength light energy yields only a single beam of light; must carefully position the single beam of light in a carefully controlled fixed relationship to the surface of the object to be imaged; and provides images which are extremely limited both in the size of the field of view as well as the quality of the image actually seen. Moreover, to employ the NSOM technique, a long period of carefully controlled raster scanning must be performed using only the single beam of light which must then travel repeatedly over large areas of sample to provide even a single image. Thus, present practical techniques of NSOM and near-field imaging as such are highly laborious, time consuming, technically stringent and demanding, and provide only single points of information which then must be carefully matched with many other individual points in order to obtain a comprehensive picture of the sample which then may be reviewed for microanalytical purposes. Accordingly, were an aperture-based optic fiber developed which would provide superresolution capabilities with a plurality of discrete subwavelength beams of light used concurrently and collectively to be developed, such an innovation would be recognized as a major advance and unexpected improvement over conventionally known light probes and near-field imaging techniques.